The goal of this project is to understand the mechanisms by which RNAs fold into biologically active structures, using the Tetrahymena self-splicing intron as a model system. This system is advantageous for studying RNA folding because the catalytic activity of the intron reflects its conformation. Self-splicing is attenuated by alternative secondary structure in the rRNA exons, and by misfolding of the catalytic core of the intron. Emphasis will be placed on understanding how long-range interactions influence the competition between alternative structures. RNA folding underlies the assembly of RNA-protein complexes and is of fundamental importance to post-transcriptional regulation of gene expression. The structures of transitions states for the early stages in tertiary folding of the Tetrahymena intron will be mapped by comparing the effects of chemical modifications on the folding equilibrium and kinetics of the P5abc subdomain. The folding kinetics of P5abc will be measured by stopped-flow fluorescence of pyrene-conjugated RNA. Fluorescent oligonucleotides that specifically bind the active or inactive conformation of the pre-rRNA will be used to measure the rate of exchange between alternative secondary structures. Together, these experiments will provide information on elementary steps of RNA folding. An array of tandem stem-loops will be engineered to systematically test long-range coupling of secondary structure elements. Fluorescent probes and chemical modification reagents will be used to directly determine the structure of the RNA, both after renaturation and following transcription. Using self-splicing and pre-mRNA splicing as reporters for RNA secondary structure in bacteria and yeast, we will investigate whether long-range structure in the pre-RNA is important for splice site recognition, or whether only local RNA and RNA-protein interactions are of functional importance. The role of transcription and flanking sequences in facilitating rapid excision of the Tetrahymena intron from its natural splice junction in the 26 S rRNA will be investigated in yeast. Mutations that stabilize misfolded intermediates will be used to probe the mechanism of folding in vivo.